Transgenic plants including a transgene consisting of a hybrid nucleic acid sequence, comprising at least one unedited mitochondrial gene fragment from higher plants and process for producing them

ABSTRACT

Hybrid nucleic acid sequences including at least the coding region of an unedited mitochondrial gene of superior plants and controlling the male fertility of plants containing said sequences, transgenic plants having such sequences and methods of production of transgenic male-sterile plants and method of restoring male-fertile plants. The nuclei of the transgenic plants contain a hybrid sequence capable of being expressed (transgene), comprising at least one coding region of an unedited mitochondrial gene of superior plants and a sequence capable of transferring the protein expressed by said coding region, to the mitochondrion, said hybrid sequence being capable of modifying the male fertility of plants having incorporated said transgene, while leaving the other phenotype characteristics of said plants unaltered.

This application is a divisional of application Ser. No. 08/505,218filed Nov. 3, 1995, now U.S. Pat. No. 5,914,447, which is a 371 ofPCT/FR94/00162 filed Feb. 15, 1994.

FIELD OF THE INVENTION

The present invention relates to hybrid nucleic acid sequences,comprising at least the coding region of an unedited mitochondrial genefrom higher plants and allowing the control of male fertility in plantscontaining the said sequences, to the transgenic plants having suchsequences, as well as to a method for producing transgenic male-sterileplants and to a method for restoring male-fertile plants.

BACKGROUND OF THE INVENTION

The control of male fertility in plants is one of the key problems forobtaining hybrids, and more particularly male-sterile lines which are ofagronomic interest especially for controlling and improving seeds.Indeed, the large scale production of hybrid seeds with controlledcharacteristics is a real challenge since many crops have both male andfemale reproductive organs (stamens and pistils). This causes a highrate of self-pollination and makes difficult the control of crossingsbetween lines for obtaining the desired hybrids.

In order to allow non-inbred crossings to be obtained which make itpossible to produce hybrid seeds having advantageous properties, theinventors have developed new transgenic male-sterile plants capable ofbeing restored and which facilitate the development of hybrid crops.

Cytoplasmic male sterility (MCS) is characterized by non-formation ofthe pollen after meiosis.

In alloplasmic systems, MCS is due to a nucleus-cytoplasmincompatibility which may occur at several levels: replication of DNA,transcription of genes, maturation of transcripts, translation orassembly of multiprotein complexes.

From the observations made on maize and petunia (Dewey R. E. et al.,Cell, 1986, 44, 439; Young E. G. et al., Cell. 1987, 50, 41), comes thehypothesis that MCS is due to a deficiency in the mitochondrialbioenergetic machinery. Indeed, MCS manifests itself by a reduction inthe ATP and NADP levels. At the cellular level, this deficiency iscorrelated with degeneration of the cells of the anther lawn, whilehaving no effect on the development of the plant.

A number of methods have been proposed in the prior art for obtainingmale-sterile plants.

There may be mentioned especially the backcrossings which lead to thesubstitution of the nuclear genome of a species by another genome andthis, in the cytoplasmic environment of the first species (alloplasmy);this substitution may also appear spontaneously in field crops. MCS canalso be obtained by protoplast fusion (Lonsdale D. M., GeneticEngineering, 1987, 6, 47).

In all these situations, the results are not reliable or reproducible;furthermore, in all cases, the manipulations are long, tedious and oftendifficult to control.

Male-sterile plants have also been obtained by transgenosis, with theaid of a gene encoding an RNAse, under the control of an anther-specificpromoter (Mariani C. et al., Nature, 1990, 347, 737). This transgene,when expressed, has a toxic effect on the cell insofar as the endogenosRNAs are degraded, thereby causing cell death.

Another system, which also introduces a new artificial and destructivefunction, has been described by Worrall D. et al., (The Plant Cell,1992, 4, 759-771) (callase system) and has the same disadvantages as theRNAse system.

Other methodologies have also been proposed forobtaining male-sterileplants; there may be mentioned especially the techniques which takeadvantage of the disruption of certain metabolic pathways (Van de MeerI. M. et al., The Plant Cell, 1992, 4, 253-262) (expression of achalcone synthase antisense gene) or the techniques involving asymmetricsomatic hybridization (Melchers C. et al., Proc. Natl. Acad. Sci. USA,1992, 89, 6832-6836) to bring into contact, as in alloplasmicmale-sterile lines, the cytoplasm of a donor individual and the nucleusof a recipient partner. The latter two processes have the majordisadvantage of being highly unpredictable as regards the desiredobjective, namely the obtaining of male-sterile plants which makes itpossible to control reproduction in these plants.

SUMMARY OF THE INVENTION

The Applicant consequently set itself the objective of obtainingtransgenic male-sterile plants in a controlled, reliable andreproducible manner which are capable of being used in agronomicprogrammes of seed improvement.

The subject of the present invention is transgenic plants having intheir nuclei an expressible hybrid sequence (transgene) comprising atleast one coding region of an unedited mitochondrial gene from higherplants and a sequence capable of transferring the protein expressed bythe said coding region to the mitochondrion, which plants arecharacterized in that:

the coding regions of the unedited mitochondrial genes are chosen fromamong the genes encoding a protein of the ATP synthase complex which arechosen from among the wheat ATP9 gene fragment, of the following formulaI:

(Seq ID No: 7) ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACAATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATTCAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATTTTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTTCTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA

or the ATP6 gene, or from among the genes encoding a protein of therespiratory chain which are chosen from among the genes for subunits 1to 7 of NAD dehydrogenase, the gene for apocytochrome b and the genesfor subunits I, II or III of cytochrome oxidase and

the sequence capable of transferring the said expressed protein to themitochondrion is selected from the group consisting of the fragmentsencoding yeast tryptophanyl tRNA synthetase (SCHMITZ, U. K. et al.,1989, The Plant Cell, 1, 783-791), and the .beta. subunit of Nicotianaplumbaginifolia ATPase (BOUTRY et al., 1987, Nature, 328:340-342), andthe maize ATP/ADP translocator (BATHGATE et al., 1989, Eur. J. Biochem.,183:303-310) or a 303 base pair EcoRI/KpnI fragment including codons 1to 62 of subunit IV of yeast cytochrome oxidase (MAARSE et al., 1984,EMBO J., 3, 2831-2837),

which hybrid sequence is capable of modifying male fertility in plantshaving incorporated the said transgene while not modifying the otherphenotypic characteristics of the said plants.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts plasmid pEA903.

FIG. 2 depicts plasmid pEA904.

FIG. 3 depicts plasmid pH1.

FIG. 4 depicts plasmid pH5.

FIG. 5 depicts plasmid pH2.

FIG. 6 depicts plasmid pH4.

FIGS. 7A-7C depict flowers from transgenic plants (7A1) and normalplants (7A2) and pollen grains from transgenic plants (7B) and fromnormal plants (7C).

FIGS. 8A-8C depict analysis of transgenes (8C) by PCR (8A) and analysisof the poly A+ transcripts of transgenic plants by Northernhybridization (8B).

FIG. 9 depicts analysis of total RNA from transformed plants H2 and H5and from control plants.

FIG. 10 depicts plasmid pGEX/coxIV.

FIG. 11 depicts the intracellular localization of transgenic proteins byimmunoblotting.

FIG. 12 depicts the Presequence of COX IV-ATP 9 (unedited).

FIG. 13 depicts the Presequence COX IV-ATP 9 (edited).

DETAILED DESCRIPTION OF THE INVENTION

According to an advantageous embodiment of the said transgenic plants,the said hybrid nucleic acid sequence comprises the coding region offormula I of the gene encoding the unedited form of wheat ATP9, withwhich is associated as transfer sequence, codons 1 to 62 of thepresequence of subunit IV of the yeast cytochrome oxidase (cox IV) (SEQID No. 1).

According to another advantageous embodiment of the said transgenicplants, the said hybrid nucleic acid sequence comprises the fragment ofthe region encoding the unedited form of wheat ATP6, of the followingformula II:

(SEQ ID NO: 8) ATG GAT AAT TTT ATC CAG AAT CTG CCT GGT GCC TAC CCG GAAACC CCA TTG GAT CAA TTT GCC ATT ATC CCA ATA ATT GAT CTT CAT GTG GGC AACTTT TAT TTA TCA TTT ACA AAT GAA GTC TTG TAT ATG CTG CTC ACT GTC GTT TTGGTC GTT TTT CTT TTT TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCAAAT GCA TGG CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AAC CTG GTAAAC GAA CAA ATA GGT GGT CTT TCC GGA AAT GTG AAA CAA AAG TTT TTC CCT CGCATC TCG GTC ACT TTT ACT TTT TCG TTA TTT CGT AAT CCC CAG GGT ATG ATA CCCTTT AGC TTC ACA GTG ACA AGT CAT TTT CTC ATT ACT TTG GCT CTT TCA TTT TCCATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA AGA CAT GGG CTT CAT TTT TTTAGC TTC TTA TTA CCT GCG GGA GTC CCA CTG CCG TTA GCA CCT TTC TTA GTA CTCCTT GAG CTA ATC TCT TAT TGT TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTTGCT AAT ATG ATG GCC GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTT GCT TGGACT ATG CTA TTT CTG AAT AAT ATT TTC TAT TTC ATA GGA GAT CTT GGT CCC TTATTT ATA GTT CTA GCA TTA ACC GGT CTG GAA TTA GGT GTA GCT ATA TCA CAA GCTCAT GTT TCT ACG ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA AAT CTC CATCAA AAT GAG TCA TTT CAT AAT TGA,

with which is associated as transfer sequence, codons 1 to 62 of thepresequence of subunit IV of yeast cytochrome oxidase (cox IV) (SEQ IDNo. 3).

According to another advantageous embodiment of the said transgenicplants, the said hybrid nucleic acid sequence comprises the fragment ofthe region encoding the unedited form of cox II of the following formulaIII:

(SEQ ID NO: 9) ATG ATT CTT CGT TCA TTA TCA TGT CGA TTC TTC ACA ATC GCTCTT TGT GAT GCT GCG GAA CCA TGG CAA TTA GGA TCT CAA GAC GCA GCA ACA CCTATG ATG CAA GGA ATC ATT GAC TTA CAT CAC GAT ATC TTT TTC TTC CTC ATT CTTATT TTG GTT TTC GTA TCA CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAGCAA ACT AAT CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA ATT ATTCGG ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT GCT ATA CCA TCG TTT GCTCTG TTA TAC TCA ATG GAC GGG GTA TTA GTA GAT CCA GCC ATT ACT ATC AAA GCTATT GGA CAT CAA TGG TAT CGG ACT TAT GAG TAT TCG GAC TAT AAC AGT TCC GATGAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT CCA GAA GAT GAT CCA GAA TTGGGT CAA TCA CGT TTA TTA GAA GTT GAC AAT AGA GTG GTT GTA CCA GCC AAA ACTCAT CTA CGT ATG ATT GTA ACA CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCTTCC TCA GGT GTC AAA TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT ACC TTC ATCTCG GTA CAA CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GAG ATT CGT GGA ACTAAT CAT GCC TTT ACG CCT ATC GTC GTA GAA GCA GTG ACT TTG AAA GAT TAT GCGGAT TGG GTA TCC AAT GAA TTA ATC CTC CAA ACC AAC TAA,

with which is associated as transfer sequence, codons 1 to 62 of thepresequence of subunit IV of yeast cytochrome oxidase (cox IV) (SEQ IDNo. 5).

The plants having incorporated the transgene in accordance with theinvention (transgenic plants) are generally selected from plants whichare of agronomic, medical or industrial interest. More precisely, anytransformable and regenerable plant can constitute the raw material forobtaining a transgenic plant in accordance with the invention.

For the purposes of the present invention, transformable is understoodto mean any plant having the possibility of integrating a gene at thenuclear level in a manner which is stable and transmissible to itsdirect progeny.

Also for the purposes of the present invention, regenerable isunderstood to mean any plant having the capacity to produce neoformedplants (neoformation or micropropagation).

In a nonlimiting manner, the following plants can be subjected totransformation in accordance with the invention:

tobacco, rape, sunflower, soya bean, tomato, potato, melon, carrot,pepper, chicory, clover, lupin, bean, pea, maize, wheat, rye, oat,barley, rice, millet, citrus, cotton.

The plants, from which the unedited mitochondrial genes are obtained,are selected such that the changes in nucleotides (process calledediting) between the unedited sequence and the edited sequence aresubstantial: at least 8 modified codons, and preferably at least 10modified codons.

Preferably, the unedited mitochondrial genes are obtained, in anonlimiting manner, from wheat, tobacco, petunia or potato.

Yeast presequences are in particular functional in the import ofproteins into the mitochondrion in plants.

In accordance with the invention, the plant from which the said uneditedmitochondrial gene is obtained and the plant which incorporated thetransgene may be identical or different.

Surprisingly, the plants transformed by such a sequence have, in atleast 50% of them, a male-sterile phenotype, while having no otherdisruptions as regards the development of the plant.

Also surprisingly, such transgenic plants make it possible to control,in a reliable and reproducible manner, the natural process of MCS,especially by avoiding self-pollination, without introducing new,artificial and destructive functions into the latter, as is the caseespecially in the systems described by Mariani et al. (RNAse system) orby WORRALL D. et al. (callase system).

The subject of the present invention is also a hybrid nucleic acidsequence, comprising at least the coding region of an uneditedmitochondrial gene from higher plants, with which is associated asequence capable of transferring the protein expressed by the saidcoding region to the mitochondrion, characterized in that:

the coding regions of the unedited mitochondrial genes are chosen fromamong the genes encoding a protein of the ATP synthase complex which arechosen from among the wheat ATP9 gene fragment, of the following formulaI:

(SEQ ID NO: 7) ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACAATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC AGT TCT TTG ATTCAT TCC GTG GCG CGA AAT CCA TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC ATTTTG GGC TTT GCT CTC ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTTCTG ATC TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA

or the ATP6 gene, or from among the genes encoding a protein of therespiratory chain, which are chosen from among the genes for subunits 1to 7 of NAD dehydrogenase, for apocytochrome b and for subunits I, II orIII of cytochrome oxidase, and

the nucleic sequence capable of transferring the said expressed proteinto the mitochondrion is selected from the group consisting of thefragments encoding yeast tryptophanyl tRNA synthetase, the f subunit ofNicotiana plumbaginifolia ATPase, the maize ATP/ADP translocator and a303 base pair EcoRI/KpnI fragment including codons 1 to 62 of subunit IVof yeast cytochrome oxidase, which hybrid sequence is capable ofmodifying male fertility in plants having incorporated it.

According to an advantageous embodiment of the said hybrid nucleic acidsequence, it comprises the coding region of formula I of the geneencoding the unedited form of wheat ATP9, with which is associated astransfer sequence, codons 1 to 62 of the presequence of subunit IV ofthe yeast cytochrome oxidase (cox IV) (SEQ ID No. 1).

According to another advantageous embodiment of the said hybrid nucleicacid sequence, it comprises the fragment of the region encoding theunedited form of wheat ATP6, of formula II above, with which isassociated as transfer sequence, codons 1 to 62 of the presequence ofsubunit IV of yeast cytochrome oxidase (cox IV) (SEQ ID No. 3).

According to another advantageous embodiment of the said hybrid nucleicacid sequence, it comprises the fragment of the region encoding theunedited form of cox II of formula III above, with which is associatedas transfer sequence, codons 1 to 62 of the presequence of subunit IV ofyeast cytochrome oxidase (cox IV) (SEQ ID No. 5).

The subject of the present invention is also a plasmid, characterized inthat it includes a hybrid nucleic acid sequence in accordance with theinvention, associated with a promoter chosen from the promoters whichare constitutively expressed and the promoters which are expressed inthe anthers and with a suitable terminator.

According to an advantageous embodiment of the said plasmid, itcomprises the 35S promoter and the terminator of the CaMV VI gene.According to another advantageous embodiment of the said plasmid, itcomprises in addition at least one marker gene, especially, and in anonlimiting manner, a gene for resistance to an antibiotic, andpreferably the gene for resistance to hygromycin.

In accordance with the invention, the transgenic plants, as definedabove, are capable of being obtained by means of a process for producingtransgenic plants which comprises, for the transformation of theselective higher plant, the introduction of at least one copy of thehybrid nucleic sequence as defined above, into a recipient plant, bymeans of a plasmid containing the said sequence, as defined above.

Such a transformation can advantageously be obtained by one of thefollowing methods: protoplast transformation, agrotransformation,microinjection, biolistic.

The subject of the present invention is also a process for inhibitingthe production of pollen in higher plants, characterized in that itcomprises the following steps:

(a) inserting a hybrid nucleic acid sequence, as defined above, into theselected plants, by any appropriate means;

(b) regenerating and culturing the transgenic plants obtained in (a);and

(c) measuring the production and the viability of the pollen (test ofgermination in particular).

Also surprisingly, the male function of the said transgenic male-sterileplants, in accordance with the invention, can be restored by crossingthe said transgenic male-sterile plants with transgenic plantscomprising in their nuclei a so-called antisense hybrid nucleic acidsequence, that is to say including at least the same coding region ofunedited plant mitochrondrial gene as that included in the saidtransgenic male-sterile plants, in the reverse direction.

The subject of the present invention is also a process for restoringmale-fertile plants, from transgenic male-sterile plants, in accordancewith the invention, characterized in that it comprises the followingsteps:

(1) transforming the selected higher plant by introducing at least onecopy of the hybrid nucleic sequence as defined above, into a recipientplant, by means of a plasmid containing the said sequence, in order toobtain transgenic male-sterile plants (TMSP); transforming the samehigher plant as in (1), by introducing at least one copy of an antisensehybrid nucleic sequence, including at least the same coding region ofthe unedited plant mitochondrial gene as that included in the saidtransgenic male-sterile plants obtained in (1), into a recipient plant,by means of a plasmid containing the said sequence, in order to obtaintransgenic male-fertile plants (TMFP); crossing the transgenicmale-sterile plants obtained in (1) and the male-fertile plants obtainedin (2), in order to obtain vigorous hybrids whose male fertility hasbeen restored and which have preselected characteristics. The subject ofthe present invention is also plasmids including an antisense hybridsequence, as defined above, associated with a promoter chosen from amongthe constitutive promoters and the promoters specific for the anthersand also associated with a suitable terminator.

In addition to the preceding arrangements, the invention also comprisesother arrangements, which will emerge from the description below, whichrefers to exemplary embodiments of the process which is the subject ofthe present invention.

It should be understood, however, that these examples are given solelyby way of illustration of the subject of the invention and do not in anymanner constitute a limitation thereto.

EXAMPLE 1

Construction of a Chimeric Gene in Accordance with the Invention CoxIV-ATP9 (SEQ ID No. 1)

The sequences encoding ATP9 are obtained from a cDNA corresponding tothe edited and unedited forms of wheat mitochondrial mRNA.

ATP9 is fused with a 303 base pair EcoRI/KpnI fragment from a plasmidcalled 19.4 (MAARSE et al., EMBO J., 1984, 3, 2831-2837), includingcodons 1 to 62 of subunit IV (cox IV) of yeast cytochrome oxidase.

The resulting fragment, obtained after digestion with the enzyme HincIIis ligated at the level of the SmaI restriction site of the plasmidpDH51 (PIETRZAK et al., 1986, Nucleic Acids Res., 14:5857-5858). Thehygromycin resistance gene is inserted at the level of the Hind III siteof the plasmid pDH51, of the plasmid pEA903 (edited form of ATP9,FIG. 1) and of the plasmid pEA904 (unedited form of ATP9, FIG. 2) givingrise to the plasmids pH1 (FIG. 3), pH5 (FIG. 4) and pH2 (FIG. 5)respectively. The plasmid pH4 (FIG. 6) consists of the plasmid pEA904 inwhich the coding part cox IV/ATP9 is placed in reverse orientationcompared with the plasmid pH2.

The unedited cox IV-ATP9 and edited cox IV-ATP9 sequences arerepresented in FIGS. 12 and 13.

All these genes are under the control of the CaMV 35S promoter and ofthe CaMV VI gene terminator.

The sequences in accordance with the invention can be specificallyamplified by means of the following oligonucleotide primers:

(a) 5′-CACTACGTCAATCTATAAG-3′ (SEQ ID No:10), extending from codon 3 tocodon 9 of the presequence of subunit IV of yeast cytochrome oxidase and5′-TATGCTCAACACATGAGCG-3′ (SEQ ID No:11), localized at the level of theCaMV VI gene terminator (45 base pairs upstream of the polyadenylationsignal). The ATP9 mRNA in wheat undergoes C—U nucleotide changes(process called editing), at the level of 8 codons. The consequence ofthese modifications is the change of 5 amino acids in the correspondingprotein (edited protein) and the loss, compared with the deducedsequence of the gene, of 6 residues from the C-terminal region, a losswhich is caused by the creation of a stop codon.

The unedited protein is more hydrophilic with 6 additional residues atthe C-terminal level; furthermore, this selected unedited form of ATP9constitutes a particularly advantageous model of modified proteinbecause it constitutes one element of the ATP synthase proton channeland, consequently, it is essential for the function of this complex;this fragment is also advantageous because of the small size of thecoding sequence, which facilitates handling, and the fact that ATP9 mayhave a nuclear or mitochondrial localization.

EXAMPLE 2

Production of Transgenic Male-sterile Plants

Both the plasmid constructs in accordance with the invention (seeExample 1, plasmid pH2) and the control constructs (plasmid pH1) and theconstructs corresponding to the edited form of ATP9 (plasmid pH5) areused for the transformation of protoplasts of a Nicotiana tabacum cv.Petit Havana line, called SR1.

Transformation of the Protoplasts

The protoplasts used for the transformation are isolated from the leavesof Nicotiana tabacum SR1 plants, cultivated under axenic conditions andone month old. The young leaves are removed, the central vein eliminatedand the leaves are cut into thin slices. The fragments are thenincubated in the dark at 26° C., overnight, in an enzymatic solutionconsisting of K3 medium (NAGY and MALIGA, 1976) supplemented with R10Onozuka cellulase (1.2%), R10 Onozuka macerozyme (0.4%) and Flukadriselase (0.1%) (pH 5.6). Before theharvest, the enzymatic solution isdiluted with a 0.6M sucrose solution, 0.1% (w/v) MES (pH 5.6) in therespective proportions 2v/1v. The protoplasts are separated from theundigested tissues by filtration through a 100 μm sieve. The suspensionis covered with a W5 solution (MENCZEL et al., Theor. Appl. Genetics,1981, 59:191-195) being careful not to mix the liquid phases. Aftercentrifuging at 600 rpm for 10 min, the protoplasts are assembled in theform of a band at the interface between the W5 solution and theenzymatic solution. They are carefully collected and washed twice withthe W5 solution in order to remove traces of enzymes. The protoplastsare placed in a cold chamber at 4-6° C. for 1-2 hours. After anothercentrifugation at 750 rpm for 5 min, they are resuspended in amannitol/magnesium solution (0.5M Merck mannitol; 1.5 mM Prolabo MgCl₂6H₂O, 0.1% Sigma MES, pH 5.6) and their concentration is adjusted to1.6×10⁶ protoplasts/ml. The protoplasts are subjected to a heat shock at45° C. for 5 minutes.

After returning to room temperature, 300 μl of protoplast suspension(5×10⁵ protoplasts) are distributed in a 12 ml conical tube. Next, 20 μgof plasmid pH2 (or of plasmid pH4), depending on the transgenic plantwhich it is desired to obtain, 300 μl of a solution of PEG 4000>40%(w/v) Merck PEG 4000; 0.4M Merck mannitol; Merck Ca(NO₃)₂ 4H₂O; pH 8(solution sterilized by filtration on 0.45 μm) and 60 μg of calf thymusDNA as carrier DNA, are added to the protoplast suspension. The mixtureis incubated at room temperature for 25-30 minutes and gently stirredfrom time to time. The transformation suspension is then graduallydiluted by adding, in small portions, 10 ml of W5 over a period of 10minutes. The protoplasts are recovered by centrifugation and taken up in1 ml of K3 medium.

Culture of the Protoplasts and Regeneration of Plants

The protoplasts are cultured in an amount of 5×10⁴ protoplasts/ml, in 3ml of a mixture of K3 and H medium (KAO and MICHAYLUK, 1975) in a 1:1(v/v) proportion, solidified with agarose (0.8%). The resulting coloniesare gradually cultured in the presence of hygromycin selection agent at20 mg/l, in A50m medium (A medium containing 50 g/l mannitol) (CABOCHE,1980) for the first month, and then on A30m medium (the A mediumcontaining 30 g/l mannitol) for the second month, and finally on A-mmedium (A medium without mannitol), medium containing 40 mg/ml ofhygromycin, during the third month. For the regeneration, the calli aretransferred onto the AR medium. The AR medium is the A medium containingonly 20 g/l sucrose as carbohydrate source and 0.25 mg/l BAP as growthhormone. The plantlets derived from the calli are cultured on T medium(NITCH and NITCH, 1969). The MSoo medium is used for maintaining theplants.

EXAMPLE 3

Phenotypic Analysis of the Transgenic Plants Obtained

The sizes of the 14-week old plants obtained in accordance with Example2 are specified in Table I below

TABLE I Fertility of the plant¹ Number of F F/S S Groove Number Linesplants tested (%) (%) (%) (cm) of nodes Seeds² (mg) SR1 1 100 0 0 87.024 109 ± 36 H1 3 100 0 0 120 ± 6  19 ± 1 108 ± 14 H2 16 50 — — — — 100 ±32 — 19 — 103 ± 26  19 ± 2  25 ± 17 — — 31 — — 0 H5 9 100 0 0 92 ± 23 23± 5  94 ± 28 ¹F = fertile, F/S = semifertile, S = malesterile ²meanvalue of production of seed per capsule after selfpollination H1 line =transgenic plants obtained with the plasmid pH1, H2 line = transgenicplants obtained with the plasmid pH2, H5 line = transgenic plantsobtained with the plasmid pH5, Control line SR1 (nontransformed plant).

The size of the plants is not significantly different from that of thenontransformed SR1 lines. The mean number of nodes is similar in thethree different transgenic lines (19 to 24 nodes per plant).

Apparently, there is no change in the function of the vegetativemeristems in the differentiation of the nodes and of the leaves of thetransgenic plants.

Flowering in the H1, H2 and H5 lines is induced to 14 weeks aftertransplantation. The flowers from the transgenic plants are similar inshape and in colour to those of the SR1 flowers (red-pink petals andanthers in each flower). The male-sterile plants have white antherscontaining few or no pollen grains (FIGS. 7A1 and 7B), whereas thefertile plants have yellow-white anthers with normal pollen grains(FIGS. 7A2 and 7C). There is no difference in the shape and in thecolour of the pistil between the male-sterile and male-fertile plants.

EXAMPLE 4

Analysis of the Fertility of the Transgenic Plants

The transformants H1 and H5 produce fertile plants, whereas thetransformants H2 have fertility, semi-fertility or sterilitycharacteristics which are defined on the basis of germination of thepollen or by the reaction with fluorescein diacetate.

In the transgenic fertile plants, the viability of the pollen is between31 and 75%, close to the values found in the SR1 control line; in thesemifertile plants, the viability of the pollen is about 10 to 20%; inthe male-sterile plants, the viability is generally less than 2%.

The fertility of the plants is also determined by the production ofseeds after self-pollination or backcrossing. The results are alsoillustrated in Table I above.

The H1 and H5 lines have a mean seed production of 100 mg/capsule,comparable with that of the SR1 control lines (110 mg/capsule). The H2lines which correspond to sterile plants produce no seed, thesemifertile plants produce between 12 and 50 mg/capsule, the fertileplants produce on average 100 mg/capsule. These values correlate wellwith the pollen viability.

The female fertility characteristic, for the sterile and semifertileplants, is determined by backcrossing with the SR1 lines as male parent.

All the male-sterile plants are fertile females and produce a normalquantity of viable seeds (63 to 92 mg/capsule), with a seed viabilityvalue greater than 77%. Thus, the sterile or semifertile character in50% of the H2 lines is due to the absence or to the very low productionof viable pollen.

The transmission of the transgenes is analysed through the geneticsegregation of the hygromycin phosphotransferase (hpt) gene in thedescendants (between 200 and 500 descendants analysed. Afterself-pollination (fertile and/or semifertile plants), the resistance tohygromycin is transmitted in most of the cases as a mendelian (mono- ordigenic) character.

After backcrossing with the SR1 parent (sterile plant), four of the fivemale-sterile plants inherit the character for hygromycin resistance as adigenic mendelian character, this expressing two active loci.

These analyses show that the sterile plants are only affected inrelation to the production of pollen, since they are fertile females andproduce a quantity of seeds per fruit (100 to 150 mg) comparable or evengreater than that of the controls.

EXAMPLE 5

Molecular Analysis of the Transformants

In order to demonstrate the presence and the transcription of the ATP9transgene, the analysis of the transcription products is performed bySouthern and Northern type hybridization. The total DNA is isolated fromthe (sterile, semifertile and fertile) H2 lines and the H5 lines.Moreover, the chimeric gene is analysed by PCR amplification.

Methods used

The total DNA is isolated from 10 g of leaf tissue essentially asdescribed in SAGHAL-MAROOF M. A. et al., 1984. Proc. Natl. Acad. Sci.USA, 81, 8014-8018. 1 μg of DNA is amplified in a final volume of 100μl, using 0.5 unit of Taq polymerase, 0.18 mM dNTPs and 100 pmol of eachof the primers. The primers used re those specified in Example 1. Theuse of these primers excludes the amplification of the endogenous ATP9(see FIG. 8C).

The denaturation step is performed at 95° C. for 1 min, thehybridization step is performed for 2 min at 52° C. and thepolymerization step is performed for 1 min at 72° C.

25 cycles are performed, the samples are subjected to electrophoresis ona 1.5% agarose gel and transferred onto a Hybond-N⁺ membrane (Amersham),as described in SAGHAL-MAROOF M. A. al. (reference cited). The filtersare prehybridized at 42° C. in 50% deionized formamide, 5×SSC,8×Denhardt and 0.5% SDS. The filters are hybridized with the 300 basepair coding sequence of ATP9, a 32P-labelled EcoRI/HindIII fragment.

A band (corresponding to a product comprising 700 base pairs) isobserved in most of the H2 and H5 lines as expected. FIG. 8A shows theresults obtained with the H2.2 and H2.16 DNA derived from male-sterileplants (lanes 1 and 2) and with fertile plants (H5.6 and H5.15 DNA,lanes 3 and 4). The DNA derived from nontransformed plants SR1 gives nosignal (lane 5).

The total RNA from the SR1, H2 and H5 lines is extracted, from theleaves, as follows: 5 g of leaves are cryo ground; then a firstextraction is performed using the frozen powder, with 5 ml of a phenol;chloroform; isoamyl alcohol mixture (25:24:1; v:v:v) and 5 ml ofTNES+DTT (0.1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% SDS and 2mM dithiothreitol); a second extraction is then performed, using theaqueous phase, twice with an equal volume of chloroform and isoamylalcohol (24:1; v:v) and the RNA is precipitated with an equal volume of4 M lithium chloride at 0° C. overnight.

The RNAs are dissolved in DEPC-treated water. The RNA concentration ismeasured by the optical density (OD) at 260 nm. The poly(A)⁺ RNAs arepurified by oligo(dT)-cellulose affinity chromatography. 20 μg of totalRNA and 1 μg of poly(A)⁺ RNA are subjected to electrophoresis on 1.5%agarose gel, formaldehyde/formamide buffer, and then transferred ontoHybond-N.sup.+ nylon membranes. The hybridizations with the ATP9 probeare performed as described above.

A 0.48 kb band is obtained with the SR1 control lines (FIG. 8B, lane 1).This band is present in all the lines and corresponds to themitochondrial endogenous mRNA.

An additional transcript, corresponding to a 0.98 kb band is presentonly in the transformed plants. As illustrated in FIG. 8B, thesemolecules can be separated from the endogenous mRNA byoligo(dT)-cellulose chromatography, confirming its cytoplasmic origin.

FIG. 8B, (lanes 3 and 4), shows the results obtained with themale-sterile plants H2.2 and H2.16 and with the fertile plants H5.6 andH5.15, (lanes 5 and 6). The 0.98 kb transcript is absent from thenontransformed controls (lane 2).

In parallel, by the PCR technique for cDNA, it is possible to obtaintranscripts derived from the transgene by virtue of the sequences addedduring the in vitro manipulation such as the presequence regionsobtained from the yeast (cox IV) and the CaMV termination region.Furthermore, only the 0.98 kb transcript hybridizes with a probeobtained from the cox. IV sequence fused with ATP9.

EXAMPLE 6

Analysis of the Production of the Chimeric Protein

In order to understand if the transgenes affect the expression of theendogenous mitochondrial ATP9 gene, the total RNA from the transformedplants H2 and H5 as well as from the control plants was hybridized witha specific mitochondrial probe.

As shown in FIG. 9, no substantial difference is observed when thetransgene is edited or unedited and the labelling is similar to that ofthe control.

The production of the transgenic protein is analysed by immunoblottingof the mitochondrial and cytosolic extracts. Antibodies directed againstfragments 21 to 54 of the presequence part of yeast cox IV, which arepart of the transgene, are obtained in rabbits.

The procedure is carried out as follows: a XbaI/KpnI fragment containingcodons 21 to 54 of yeast cox IV is isolated from the abovementionedplasmid 19.4.

This fragment is ligated to the plasmid pGEX-A (FIG. 10) in phase withthe coding sequence of glutathione S-transferase, under the control ofthe β-galactosidase promoter.

The fusion protein is induced after transformation of E. coli DH5A cellsby IPTG. These cells produce about 80 mg of protein per liter ofculture.

The fused protein is purified from an E. coli extract by affinitychromatography on a glutathione agarose column. The protein eluted byglutathione is obtained with a purity level of the order of 95%. Thefusion protein is used as antigen to produce anti-cox IV antibodies inrabbits.

Greenhouse plant leaves are used for cell fractionation. 100 μg ofcytosolic and mitochondrial proteins are fractionated by urea/SDS-PAGE.The immunoreaction is performed using an anti-cox IV antiserum diluted1/500th according to the DARLEY-USMAR et al. method >1987, Mitochondria,a practical approach, eds DARLEY-USMAR, (IRL Press Ltd.) pp. 113-152!.The proteins from transgenic plants carrying the male-sterile phenotypeare revealed by peroxidase-conjugated anti-rabbit IgG antibodies.

No signal is observed either with the mitochondrial fraction (FIG. 11B,lane 1), or with the cytosolic fraction from the nontransformed SR1line.

The mitochondrial fraction of the H2.2 male-sterile and H5.15 fertileplants (FIG. 11B, lanes 2 and 4 respectively) show a 12 kDa bandcorresponding to the expected size for the protein (see FIG. 11A, whichspecifies the structure of the 15 kDa precursor and the 12 kDa importedprotein).

The cytosolic proteins from these lines (FIG. 11B, lanes 3 and 5) showtwo bands, one at 15 kDa, the expected size for the chimeric precursorpolypeptide, and the other at 14 kDa. The nature of this latterpolypeptide remains to be determined; it is probably a degradationproduct of the 15 kDa precursor.

The protein associated with the mitochondrial fraction of the H5.15 line(FIG. 11, lane 4) migrates roughly to the same position as themitochondrial protein H2.2, but slightly downstream. This difference isdue to the fact that the chimeric genes differ in the position of theirstop codon. Indeed, as already specified above, the edited protein has 6residues less than the unedited protein due to the generation of a stopcodon during the editing of the RNA.

EXAMPLE 7

Study of the Respiration of the Mitochondria from the Transgenic Plants

The effect of the transgene at the subcellular level should result in adysfunction of the respiratory function of the mitochondrion. Analysisof the respiration of the nonchlorophyllian plants of the transgenicplants was performed.

The determination of the respiration rates of the nonchlorophyllianorgans (roots), in the presence or in the absence of decouplers, iscarried out by analysing the consumption of oxygen by means of a Clarkelectrode. More detailed studies were performed on mitochondria purifiedby differential centrifugation and on a Ficoll gradient. The effect ofdecouplers on respiration and the ADP/O ratios were determined onmitochondria derived from male-sterile lines and compared with thetransformed or wild-type control plants.

These different measurements show that the mitochondrial function isreduced in the male-sterile plants compared with the nontransformed ortransformed control with the plasmid pH5. This situation is similar tothat encountered in the natural male-sterile plants.

It stems from the above that the expression in the transgenic tobaccoplants of a DNA sequence encoding unedited wheat mitochondrial ATP9 hasno effect on most of the phenotypic characters of the transformedplants, except for the appearance of male sterility.

Indeed, the size, the growth rate, the number of nodes, the shape andthe size of the leaves and of the flowers are similar in the transgenicplants and in the control plants. However, significant effects areobserved in the male reproductive organs when the wheat ATP9 sequence,in its unedited form, is expressed in tobacco plants.

Indeed, the transformation experiments performed with the plasmid pH2lead to the production of many plants (50%) modified in relation totheir fertility. Approximately 19% are semifertile and 31% arecompletely sterile.

All the semifertile and sterile H2 lines express the transgene in thepolyadenylated mRNA form. The fertile H2 lines do not have the 0.98 kbtranscript, even when the transgene is detected after PCR amplification,thereby indicating that the transgene is inactive in this latter case.

Some results also show, unexpectedly, that the male-sterile phenotype iscorrelated only with the presence of unedited ATP9 sequence whereas thetransformants obtained with the edited ATP9 form are all fertile.

In all cases, the sterile plants are only male-sterile plants and can bepollenated with a foreign pollen, thereby reflecting a normal femalefertility.

EXAMPLE 8

Production of Transgenic Plants Having an Antisense Hybrid Sequence inAccordance with the Invention

The procedure is carried out as in Example 2, the transformation ofprotoplasts being however performed by means of the plasmids pH4.

By crossing these male-fertile plants with the male-sterile transgenicplants in accordance with the invention, noninbred male-fertile hybridsare obtained.

EXAMPLE 9

Construction of a Chimeric Gene in Accordance with the Invention CoxIV-ATP6 (SEQ ID No. 3)

The sequences encoding ATP6 are obtained from a cDNA corresponding tothe edited and unedited forms of wheat mitochondrial mRNA.

The unedited ATP6 fragment selected has the sequence of formula IIdefined above and is fused with the yeast transfer sequence cox IV asdefined above.

The resulting fragment is similar to that obtained in Example 1.

The ATP6 mRNA in wheat undergoes nucleotide changes (editing) at thelevel of 12 codons. The consequence of the modifications is the changeof 11 amino acids and the loss, compared with the deduced sequence ofthe gene, of 7 residues, from the C-terminal region, a loss caused bythe creation of a stop codon.

EXAMPLE 10

Construction of a Chimeric Gene in Accordance with the Invention coxIV-cox II (SEQ ID No.5)

The sequences encoding cox II are obtained from a cDNA corresponding tothe edited and unedited forms of wheat mitochondrial mRNA.

The fragment of the unedited cox II gene has the sequence of formula IIIdefined above and is fused with the yeast transfer sequence cox IV asdefined above.

The resulting fragment is similar to that obtained in Example 1.

The mRNA in wheat undergoes nucleotide changes (editing) in 16 codons.The consequence of the modifications is the change of 16 amino acidscompared with the deduced sequence of the cox II gene.

As evident from the above, the invention is riot in the least limited tothe implementations, embodiments and applications which have just beendescribed more explicitly; on the contrary, it embraces all the variantswhich may occur to a specialist in this field without departing from theframework or the scope of the present invention.

13 1 568 DNA WHEAT ATP9 CDS (99)..(524) 1 gtcaacgtat tcttctccctgaagaaacag tatactaaca atactcaccc atttcgattt 60 tgatgttgcc atacaaatagataacaagca caagcaca atg ctt tca cta cgt caa 116 Met Leu Ser Leu Arg Gln1 5 tct ata aga ttt ttc aag cca gcc aca aga act ttg tgt agc tct aga 164Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser Ser Arg 10 15 20tat ctg ctt cag caa aaa ccc gtg gtg aaa act gcc caa aac tta gca 212 TyrLeu Leu Gln Gln Lys Pro Val Val Lys Thr Ala Gln Asn Leu Ala 25 30 35 gaagtt aat ggt cca gaa act ttg att ggt cct ggt gct aaa gag ggt 260 Glu ValAsn Gly Pro Glu Thr Leu Ile Gly Pro Gly Ala Lys Glu Gly 40 45 50 acc cgggga tcc tct aga gtc gag atg tta gaa ggt gct aaa tca ata 308 Thr Arg GlySer Ser Arg Val Glu Met Leu Glu Gly Ala Lys Ser Ile 55 60 65 70 ggt gccgga gct gct aca att gct tta gcc gga gct gct gtc ggt att 356 Gly Ala GlyAla Ala Thr Ile Ala Leu Ala Gly Ala Ala Val Gly Ile 75 80 85 gga aac gtcctc agt tct ttg att act tcc gtg gcg cga aat cca tca 404 Gly Asn Val LeuSer Ser Leu Ile Thr Ser Val Ala Arg Asn Pro Ser 90 95 100 ttg gct aaacaa tca ttt ggt tat gcc att ttg ggc ttt gct ctc acc 452 Leu Ala Lys GlnSer Phe Gly Tyr Ala Ile Leu Gly Phe Ala Leu Thr 105 110 115 gaa gct attgca ttg ttt gcc cca atg atg gcc ttt ctg atc tca ttc 500 Glu Ala Ile AlaLeu Phe Ala Pro Met Met Ala Phe Leu Ile Ser Phe 120 125 130 gtt ttc cgatcg cat aaa aag tca tgagatcaaa aaagaaatgt gtgaatgtag 554 Val Phe Arg SerHis Lys Lys Ser 135 140 ttacagatgt cgac 568 2 142 PRT WHEAT ATP9 2 MetLeu Ser Leu Arg Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gln Gln Lys Pro Val Val Lys 20 25 30Thr Ala Gln Asn Leu Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly 35 40 45Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Ser Arg Val Glu Met Leu 50 55 60Glu Gly Ala Lys Ser Ile Gly Ala Gly Ala Ala Thr Ile Ala Leu Ala 65 70 7580 Gly Ala Ala Val Gly Ile Gly Asn Val Leu Ser Ser Leu Ile Thr Ser 85 9095 Val Ala Arg Asn Pro Ser Leu Ala Lys Gln Ser Phe Gly Tyr Ala Ile 100105 110 Leu Gly Phe Ala Leu Thr Glu Ala Ile Ala Leu Phe Ala Pro Met Met115 120 125 Ala Phe Leu Ile Ser Phe Val Phe Arg Ser His Lys Lys Ser 130135 140 3 1106 DNA WHEAT ATP9 CDS (99)..(1103) 3 gtcaacgtat tcttctccctgaagaaacag tatactaaca atactcaccc atttcgattt 60 tgatgttgcc atacaaatagataacaagca caagcaca atg ctt tca cta cgt caa 116 Met Leu Ser Leu Arg Gln1 5 tct ata aga ttt ttc aag cca gcc aca aga act ttg tgt agc tct aga 164Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser Ser Arg 10 15 20tat ctg ctt cag caa aaa ccc gtg gtg aaa act gcc caa aac tta gca 212 TyrLeu Leu Gln Gln Lys Pro Val Val Lys Thr Ala Gln Asn Leu Ala 25 30 35 gaagtt aat ggt cca gaa act ttg att ggt cct ggt gct aaa gag ggt 260 Glu ValAsn Gly Pro Glu Thr Leu Ile Gly Pro Gly Ala Lys Glu Gly 40 45 50 acc cgggga tcc tct aga gtc gag atg gat aat ttt atc cag aat ctg 308 Thr Arg GlySer Ser Arg Val Glu Met Asp Asn Phe Ile Gln Asn Leu 55 60 65 70 cct ggtgcc tac ccg gaa acc cca ttg gat caa ttt gcc att atc cca 356 Pro Gly AlaTyr Pro Glu Thr Pro Leu Asp Gln Phe Ala Ile Ile Pro 75 80 85 ata att gatctt cat gtg ggc aac ttt tat tta tca ttt aca aat gaa 404 Ile Ile Asp LeuHis Val Gly Asn Phe Tyr Leu Ser Phe Thr Asn Glu 90 95 100 gtc ttg tatatg ctg ctc act gtc gtt ttg gtc gtt ttt ctt ttt ttt 452 Val Leu Tyr MetLeu Leu Thr Val Val Leu Val Val Phe Leu Phe Phe 105 110 115 gtt gtt acgaaa aag gga ggt gga aag tca gtg cca aat gca tgg cca 500 Val Val Thr LysLys Gly Gly Gly Lys Ser Val Pro Asn Ala Trp Pro 120 125 130 tcc ttg gtcgag ctt att tat gat ttc gtg ctg aac ctg gta aac gaa 548 Ser Leu Val GluLeu Ile Tyr Asp Phe Val Leu Asn Leu Val Asn Glu 135 140 145 150 caa ataggt ggt ctt tcc gga aat gtg aaa caa aag ttt ttc cct cgc 596 Gln Ile GlyGly Leu Ser Gly Asn Val Lys Gln Lys Phe Phe Pro Arg 155 160 165 atc tcggtc act ttt act ttt tcg tta ttt cgt aat ccc cag ggt atg 644 Ile Ser ValThr Phe Thr Phe Ser Leu Phe Arg Asn Pro Gln Gly Met 170 175 180 ata cccttt agc ttc aca gtg aca agt cat ttt ctc att act ttg gct 692 Ile Pro PheSer Phe Thr Val Thr Ser His Phe Leu Ile Thr Leu Ala 185 190 195 ctt tcattt tcc att ttt ata ggc att acg atc gtt gga ttt caa aga 740 Leu Ser PheSer Ile Phe Ile Gly Ile Thr Ile Val Gly Phe Gln Arg 200 205 210 cat gggctt cat ttt ttt agc ttc tta tta cct gcg gga gtc cca ctg 788 His Gly LeuHis Phe Phe Ser Phe Leu Leu Pro Ala Gly Val Pro Leu 215 220 225 230 ccgtta gca cct ttc tta gta ctc ctt gag cta atc tct ata tgt ttt 836 Pro LeuAla Pro Phe Leu Val Leu Leu Glu Leu Ile Ser Ile Cys Phe 235 240 245 cgtgca tta agc tta gga ata cgt tta ttt gct aat atg atg gcc ggt 884 Arg AlaLeu Ser Leu Gly Ile Arg Leu Phe Ala Asn Met Met Ala Gly 250 255 260 catagt tta gta aag att tta agt ggg ttt gct tgg act atg cta ttt 932 His SerLeu Val Lys Ile Leu Ser Gly Phe Ala Trp Thr Met Leu Phe 265 270 275 ctgaat aat att ttc tat ttc ata gga gat ctt ggt ccc tta ttt ata 980 Leu AsnAsn Ile Phe Tyr Phe Ile Gly Asp Leu Gly Pro Leu Phe Ile 280 285 290 gttcta gca tta acc ggt ctg gaa tta ggt gta gct ata tca caa gct 1028 Val LeuAla Leu Thr Gly Leu Glu Leu Gly Val Ala Ile Ser Gln Ala 295 300 305 310cat gtt tct acg atc tca att tgt att tac ttg aat gat gct aca aat 1076 HisVal Ser Thr Ile Ser Ile Cys Ile Tyr Leu Asn Asp Ala Thr Asn 315 320 325ctc act caa aat gag tca ttt cat aat tga 1106 Leu Thr Gln Asn Glu Ser PheHis Asn 330 335 4 335 PRT WHEAT ATP9 4 Met Leu Ser Leu Arg Gln Ser IleArg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15 Thr Leu Cys Ser Ser Arg TyrLeu Leu Gln Gln Lys Pro Val Val Lys 20 25 30 Thr Ala Gln Asn Leu Ala GluVal Asn Gly Pro Glu Thr Leu Ile Gly 35 40 45 Pro Gly Ala Lys Glu Gly ThrArg Gly Ser Ser Arg Val Glu Met Asp 50 55 60 Asn Phe Ile Gln Asn Leu ProGly Ala Tyr Pro Glu Thr Pro Leu Asp 65 70 75 80 Gln Phe Ala Ile Ile ProIle Ile Asp Leu His Val Gly Asn Phe Tyr 85 90 95 Leu Ser Phe Thr Asn GluVal Leu Tyr Met Leu Leu Thr Val Val Leu 100 105 110 Val Val Phe Leu PhePhe Val Val Thr Lys Lys Gly Gly Gly Lys Ser 115 120 125 Val Pro Asn AlaTrp Pro Ser Leu Val Glu Leu Ile Tyr Asp Phe Val 130 135 140 Leu Asn LeuVal Asn Glu Gln Ile Gly Gly Leu Ser Gly Asn Val Lys 145 150 155 160 GlnLys Phe Phe Pro Arg Ile Ser Val Thr Phe Thr Phe Ser Leu Phe 165 170 175Arg Asn Pro Gln Gly Met Ile Pro Phe Ser Phe Thr Val Thr Ser His 180 185190 Phe Leu Ile Thr Leu Ala Leu Ser Phe Ser Ile Phe Ile Gly Ile Thr 195200 205 Ile Val Gly Phe Gln Arg His Gly Leu His Phe Phe Ser Phe Leu Leu210 215 220 Pro Ala Gly Val Pro Leu Pro Leu Ala Pro Phe Leu Val Leu LeuGlu 225 230 235 240 Leu Ile Ser Ile Cys Phe Arg Ala Leu Ser Leu Gly IleArg Leu Phe 245 250 255 Ala Asn Met Met Ala Gly His Ser Leu Val Lys IleLeu Ser Gly Phe 260 265 270 Ala Trp Thr Met Leu Phe Leu Asn Asn Ile PheTyr Phe Ile Gly Asp 275 280 285 Leu Gly Pro Leu Phe Ile Val Leu Ala LeuThr Gly Leu Glu Leu Gly 290 295 300 Val Ala Ile Ser Gln Ala His Val SerThr Ile Ser Ile Cys Ile Tyr 305 310 315 320 Leu Asn Asp Ala Thr Asn LeuThr Gln Asn Glu Ser Phe His Asn 325 330 335 5 1067 DNA WHEAT ATP9 CDS(99)..(1064) 5 gtcaacgtat tcttctccct gaagaaacag tatactaaca atactcacccatttcgattt 60 tgatgttgcc atacaaatag ataacaagca caagcaca atg ctt tca ctacgt caa 116 Met Leu Ser Leu Arg Gln 1 5 tct ata aga ttt ttc aag cca gccaca aga act ttg tgt agc tct aga 164 Ser Ile Arg Phe Phe Lys Pro Ala ThrArg Thr Leu Cys Ser Ser Arg 10 15 20 tat ctg ctt cag caa aaa ccc gtg gtgaaa act gcc caa aac tta gca 212 Tyr Leu Leu Gln Gln Lys Pro Val Val LysThr Ala Gln Asn Leu Ala 25 30 35 gaa gtt aat ggt cca gaa act ttg att ggtcct ggt gct aaa gag ggt 260 Glu Val Asn Gly Pro Glu Thr Leu Ile Gly ProGly Ala Lys Glu Gly 40 45 50 acc cgg gga tcc tct aga gtc gag atg att cttcgt tca tta tca tgt 308 Thr Arg Gly Ser Ser Arg Val Glu Met Ile Leu ArgSer Leu Ser Cys 55 60 65 70 cga ttc ttc aga atc gct ctt tgt gat gct gcggaa cca tgg caa tta 356 Arg Phe Phe Arg Ile Ala Leu Cys Asp Ala Ala GluPro Trp Gln Leu 75 80 85 gga tct caa gac gca gca aca cct atg atg caa ggaatc att gac tta 404 Gly Ser Gln Asp Ala Ala Thr Pro Met Met Gln Gly IleIle Asp Leu 90 95 100 cat cac gat atc ttt ttc ttc ctc att ctt att ttggtt ttc gta tca 452 His His Asp Ile Phe Phe Phe Leu Ile Leu Ile Leu ValPhe Val Ser 105 110 115 cgg atg ttg gtt cgc gct tta tgg cat ttc aac gagcaa act aat cca 500 Arg Met Leu Val Arg Ala Leu Trp His Phe Asn Glu GlnThr Asn Pro 120 125 130 atc cca caa agg att gtt cat gga act act atg gaaatt att cgg acc 548 Ile Pro Gln Arg Ile Val His Gly Thr Thr Met Glu IleIle Arg Thr 135 140 145 150 ata ttt cca agt gtc att ctt ttg ttc att gctata cca tcg ttt gct 596 Ile Phe Pro Ser Val Ile Leu Leu Phe Ile Ala IlePro Ser Phe Ala 155 160 165 ctg tta tac tca atg gac ggg gta tta gta gatcca gcc att act atc 644 Leu Leu Tyr Ser Met Asp Gly Val Leu Val Asp ProAla Ile Thr Ile 170 175 180 aaa gct att gga cat caa tgg tat cgg act tatgag tat tcg gac tat 692 Lys Ala Ile Gly His Gln Trp Tyr Arg Thr Tyr GluTyr Ser Asp Tyr 185 190 195 aac agt tcc gat gaa cag tca ctc act ttt gacagt tat acg att cca 740 Asn Ser Ser Asp Glu Gln Ser Leu Thr Phe Asp SerTyr Thr Ile Pro 200 205 210 gaa gat gat cca gaa ttg ggt caa tca cgt ttatta gaa gtt gac aat 788 Glu Asp Asp Pro Glu Leu Gly Gln Ser Arg Leu LeuGlu Val Asp Asn 215 220 225 230 aga gtg gtt gta cca gcc aaa act cat ctacgt atg att gta aca ccc 836 Arg Val Val Val Pro Ala Lys Thr His Leu ArgMet Ile Val Thr Pro 235 240 245 gct gat gta cct cat agt tgg gct gta ccttcc tca ggt gtc aaa tgt 884 Ala Asp Val Pro His Ser Trp Ala Val Pro SerSer Gly Val Lys Cys 250 255 260 gat gct gta cct ggt cgt tca aat ctt accttc atc tcg gta caa cga 932 Asp Ala Val Pro Gly Arg Ser Asn Leu Thr PheIle Ser Val Gln Arg 265 270 275 gaa gaa gtt tca tat ggt cag tgc agt gagatt cgt gga act aat cat 980 Glu Glu Val Ser Tyr Gly Gln Cys Ser Glu IleArg Gly Thr Asn His 280 285 290 gcc ttt acg cct atc gtc gta gaa gca gtgact ttg aaa gat tat gcg 1028 Ala Phe Thr Pro Ile Val Val Glu Ala Val ThrLeu Lys Asp Tyr Ala 295 300 305 310 gat tgg gta tcc aat caa tta atc ctccaa acc aac taa 1067 Asp Trp Val Ser Asn Gln Leu Ile Leu Gln Thr Asn 315320 6 322 PRT WHEAT ATP9 6 Met Leu Ser Leu Arg Gln Ser Ile Arg Phe PheLys Pro Ala Thr Arg 1 5 10 15 Thr Leu Cys Ser Ser Arg Tyr Leu Leu GlnGln Lys Pro Val Val Lys 20 25 30 Thr Ala Gln Asn Leu Ala Glu Val Asn GlyPro Glu Thr Leu Ile Gly 35 40 45 Pro Gly Ala Lys Glu Gly Thr Arg Gly SerSer Arg Val Glu Met Ile 50 55 60 Leu Arg Ser Leu Ser Cys Arg Phe Phe ArgIle Ala Leu Cys Asp Ala 65 70 75 80 Ala Glu Pro Trp Gln Leu Gly Ser GlnAsp Ala Ala Thr Pro Met Met 85 90 95 Gln Gly Ile Ile Asp Leu His His AspIle Phe Phe Phe Leu Ile Leu 100 105 110 Ile Leu Val Phe Val Ser Arg MetLeu Val Arg Ala Leu Trp His Phe 115 120 125 Asn Glu Gln Thr Asn Pro IlePro Gln Arg Ile Val His Gly Thr Thr 130 135 140 Met Glu Ile Ile Arg ThrIle Phe Pro Ser Val Ile Leu Leu Phe Ile 145 150 155 160 Ala Ile Pro SerPhe Ala Leu Leu Tyr Ser Met Asp Gly Val Leu Val 165 170 175 Asp Pro AlaIle Thr Ile Lys Ala Ile Gly His Gln Trp Tyr Arg Thr 180 185 190 Tyr GluTyr Ser Asp Tyr Asn Ser Ser Asp Glu Gln Ser Leu Thr Phe 195 200 205 AspSer Tyr Thr Ile Pro Glu Asp Asp Pro Glu Leu Gly Gln Ser Arg 210 215 220Leu Leu Glu Val Asp Asn Arg Val Val Val Pro Ala Lys Thr His Leu 225 230235 240 Arg Met Ile Val Thr Pro Ala Asp Val Pro His Ser Trp Ala Val Pro245 250 255 Ser Ser Gly Val Lys Cys Asp Ala Val Pro Gly Arg Ser Asn LeuThr 260 265 270 Phe Ile Ser Val Gln Arg Glu Glu Val Ser Tyr Gly Gln CysSer Glu 275 280 285 Ile Arg Gly Thr Asn His Ala Phe Thr Pro Ile Val ValGlu Ala Val 290 295 300 Thr Leu Lys Asp Tyr Ala Asp Trp Val Ser Asn GlnLeu Ile Leu Gln 305 310 315 320 Thr Asn 7 243 DNA WHEAT ATP9 7atgttagaag gtgctaaatc aataggtgcc ggagctgcta caattgcttt agccggagct 60gctgtcggta ttggaaacgt cctcagttct ttgattcatt ccgtggcgcg aaatccatca 120ttggctaaac aatcatttgg ttatgccatt ttgggctttg ctctcaccga agctattgca 180ttgtttgccc caatgatggc ctttctgatc tcattcgttt tccgatcgca taaaaagtca 240tga 243 8 822 DNA WHEAT ATP9 8 atggataatt ttatccagaa tctgcctggtgcctacccgg aaaccccatt ggatcaattt 60 gccattatcc caataattga tcttcatgtgggcaactttt atttatcatt tacaaatgaa 120 gtcttgtata tgctgctcac tgtcgttttggtcgtttttc ttttttttgt tgttacgaaa 180 aagggaggta gaaagtcagt gccaaatgcatggcaatcct tggtcgagct tatttatgat 240 ttcgtgctga acctggtaaa cgaacaaataggtggtcttt ccggaaatgt gaaacaaaag 300 tttttccctc gcatctcggt cacttttactttttcgttat ttcgtaatcc ccagggtatg 360 atacccttta gcttcacagt gacaagtcattttctcatta ctttggctct ttcattttcc 420 atttttatag gcattacgat cgttggatttcaaagacatg ggcttcattt ttttagcttc 480 ttattacctg cgggagtccc actgccgttagcacctttct tagtactcct tgagctaatc 540 tcttattgtt ttcgtgcatt aagcttaggaatacgtttat ttgctaatat gatggccggt 600 catagtttag taaagatttt aagtgggtttgcttggacta tgctatttct gaataatatt 660 ttctatttca taggagatct tggtcccttatttatagttc tagcattaac cggtctggaa 720 ttaggtgtag ctatatcaca agctcatgtttctacgatct caatttgtat ttacttgaat 780 gatgctacaa atctccatca aaatgagtcatttcataatt ga 822 9 783 DNA WHEAT ATP9 9 atgattcttc gttcattatcatgtcgattc ttcacaatcg ctctttgtga tgctgcggaa 60 ccatggcaat taggatctcaagacgcagca acacctatga tgcaaggaat cattgactta 120 catcacgata tctttttcttcctcattctt attttggttt tcgtatcacg gatgttggtt 180 cgcgctttat ggcatttcaacgagcaaact aatccaatcc cacaaaggat tgttcatgga 240 actactatgg aaattattcggaccatattt ccaagtgtca ttcttttgtt cattgctata 300 ccatcgtttg ctctgttatactcaatggac ggggtattag tagatccagc cattactatc 360 aaagctattg gacatcaatggtatcggact tatgagtatt cggactataa cagttccgat 420 gaacagtcac tcacttttgacagttatacg attccagaag atgatccaga attgggtcaa 480 tcacgtttat tagaagttgacaatagagtg gttgtaccag ccaaaactca tctacgtatg 540 attgtaacac ccgctgatgtacctcatagt tgggctgtac cttcctcagg tgtcaaatgt 600 gatgctgtac ctggtcgttcaaatcttacc ttcatctcgg tacaacgaga aggagtttac 660 tatggtcagt gcagtgagattcgtggaact aatcatgcct ttacgcctat cgtcgtagaa 720 gcagtgactt tgaaagattatgcggattgg gtatccaatc aattaatcct ccaaaccaac 780 taa 783 10 19 DNA WHEATATP9 10 cactacgtca atctataag 19 11 19 DNA WHEAT ATP9 11 tatgctcaacacatgagcg 19 12 568 DNA WHEAT ATP9 CDS (99)..(506) 12 gtcaacgtattcttctccct gaagaaacag tatactaaca atactcaccc atttcgattt 60 tgatgttgccatacaaatag ataacaagca caagcaca atg ctt tca cta cgt caa 116 Met Leu SerLeu Arg Gln 1 5 tct ata aga ttt ttc aag cca gcc aca aga act ttg tgt agctct aga 164 Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg Thr Leu Cys Ser SerArg 10 15 20 tat ctg ctt cag caa aaa ccc gtg gtg aaa act gcc caa aac ttagca 212 Tyr Leu Leu Gln Gln Lys Pro Val Val Lys Thr Ala Gln Asn Leu Ala25 30 35 gaa gtt aat ggt cca gaa act ttg att ggt cct ggt gct aaa gag ggt260 Glu Val Asn Gly Pro Glu Thr Leu Ile Gly Pro Gly Ala Lys Glu Gly 4045 50 acc cgg gga tcc tct aga gtc gag atg tta gaa ggt gct aaa tta ata308 Thr Arg Gly Ser Ser Arg Val Glu Met Leu Glu Gly Ala Lys Leu Ile 5560 65 70 ggt gcc gga gct gct aca att gct tta gcc gga gct gct gtc ggt att356 Gly Ala Gly Ala Ala Thr Ile Ala Leu Ala Gly Ala Ala Val Gly Ile 7580 85 gga aac gtt ttc agt tct ttg att cat tcc gtg gcg cga aat cca tca404 Gly Asn Val Phe Ser Ser Leu Ile His Ser Val Ala Arg Asn Pro Ser 9095 100 ttc gct aaa caa tta ttt ggt tat gcc att ttg ggc ttt gct ctc acc452 Phe Ala Lys Gln Leu Phe Gly Tyr Ala Ile Leu Gly Phe Ala Leu Thr 105110 115 gaa gct att gca ttg ttt gcc cta atg atg gcc ttt ttg atc tta ttc500 Glu Ala Ile Ala Leu Phe Ala Leu Met Met Ala Phe Leu Ile Leu Phe 120125 130 gtt ttc tgatcgcata aaaagtcatg agatcaaaaa agaaatgtgt gaatgtagtt556 Val Phe 135 acagatgtcg ac 568 13 136 PRT WHEAT ATP9 13 Met Leu SerLeu Arg Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15 Thr LeuCys Ser Ser Arg Tyr Leu Leu Gln Gln Lys Pro Val Val Lys 20 25 30 Thr AlaGln Asn Leu Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gly 35 40 45 Pro GlyAla Lys Glu Gly Thr Arg Gly Ser Ser Arg Val Glu Met Leu 50 55 60 Glu GlyAla Lys Leu Ile Gly Ala Gly Ala Ala Thr Ile Ala Leu Ala 65 70 75 80 GlyAla Ala Val Gly Ile Gly Asn Val Phe Ser Ser Leu Ile His Ser 85 90 95 ValAla Arg Asn Pro Ser Phe Ala Lys Gln Leu Phe Gly Tyr Ala Ile 100 105 110Leu Gly Phe Ala Leu Thr Glu Ala Ile Ala Leu Phe Ala Leu Met Met 115 120125 Ala Phe Leu Ile Leu Phe Val Phe 130 135

What is claimed is:
 1. Transgenic plants having in their nuclei anexpressible hybrid sequence comprising at least one coding region of anunedited mitochondrial gene from higher plants and a sequence capable oftransferring the protein expressed by the said coding region to themitochondrion, wherein: the coding regions of the unedited mitochondrialgenes are selected from the group consisting of the genes encoding aprotein of the ATP synthase complex which are selected from the groupconsisting of the wheat ATP9 gene fragment of SEQ ID No:7 in uneditedform and ligated to a mitochondrial transporter sequence, wherein thehybrid nucleic acid sequence comprising the ATP 9 gene, when transformedinto a recipient plant, causes male sterility, and the wheat ATP6 genein unedited form and ligated to a mitochondrial transporter sequence,wherein the hybrid nucleic acid sequence comprising the ATP 6 gene, whentransformed into a recipient plant, causes male sterility, and thesequence capable of transferring the said expressed protein to themitochondrion is selected from the group consisting of the fragmentsencoding yeast tryptophanyl tRNA synthetase, the β subunit of Nicotianaplumbaginifolia ATPase, the maize ATP/ADP translocator and a 303 basepair EcoRI/KpnI fragment comprising codons 1 to 62 of subunit IV ofyeast cytochrome oxidase, which hybrid sequence is capable of modifyingmale fertility in plants having incorporated the said transgene whilenot modifying the other phenotypic characteristics of the said plants.2. Transgenic plants according to claim 1, wherein said hybrid nucleicacid sequence comprises the region of the gene encoding the uneditedform of wheat ATP9, of SEQ ID NO: 7, with which is associated astransfer sequence, condons 1 to 62 of the presequence of subunit IV ofthe yeast cytochrome oxidase (cox IV) (SEQ ID No. 1).
 3. Transgenicplants according to claim 1, wherein said hybrid nucleic acid sequencecomprises the fragment of the region encoding the unedited form of wheatATP6, of SEQ ID NO: 8, with which is associated as transfer sequence,codons 1 to 62 of the presequence of subunit IV of yeast cytochromeoxidase (cox IV) (SEQ ID No. 3).
 4. Transgenic plant according to claim1, wherein the plant having incorporated the said hybrid sequence istobacco.
 5. Transgenic plant according to claim 1, wherein the planthaving incorporated the said hybrid sequence is selected from the groupconsisting of rape, sunflower, soya bean, tomato, potato, melon, carrot,pepper, chicory, clover, lupin, bean, pea, maize, wheat, rye, oat,barley, rice, millet, citrus and cotton.
 6. Hybrid nucleic acidsequence, comprising at least the coding region of an uneditedmitochondrial gene from higher plants, with which is associated asequence capable of transferring the protein expressed by the saidcoding region to the mitochondrion, wherein: the coding regions of theunedited mitochondrial genes are selected from the group consisting ofthe genes encoding a protein of the ATP synthase complex which areselected from the group consisting of the wheat ATP9 gene fragment ofSeq ID No:7 in unedited form and ligated to a mitochondrial transportersequence, wherein the hybrid nucleic acid sequence comprising the ATP 9gene, when transformed into a recipient plant, causes male sterility,and the wheat ATP6 gene in unedited form and ligated to a mitochondrialtransporter sequence, wherein the hybrid nucleic acid sequencecomprising the ATP 6A gene, when transformed into a recipient plant,causes male sterility, and the nucleic sequence capable of transferringthe said expressed protein to the mitochondrion is selected from thegroup consisting of the fragments encoding yeast tryptophanyl tRNAsynthetase, the β subunit of Nicotiana plumbaginifolia ATPase, the maizeATP/ADP translocator and a 303 base pair EcoRI/KpnI fragment comprisingcodons 1 to 62 of subunit IV of yeast cytochrome oxidase, which hybridsequence is capable of modifying male fertility in plants havingincorporated it.
 7. Hybrid nucleic acid sequence according to claim 6,comprising the region of the gene encoding the unedited form of wheatATP9, of SEQ ID NO: 7, with which is associated as transfer sequence,codons 1 to 62 of the presequence of subunit IV of yeast cytochromeoxidase (cox I) (SEQ ID No. 1).
 8. Hybrid nucleic acid sequenceaccording to claim 6, comprising the fragment of the region encoding theunedited form of wheat ATP6 of SEQ ID NO: 8, with which is associated astransfer sequence, codons 1 to 62 of the presequence of subunit IV ofyeast cytochrome oxidase (cox IV) (SEQ ID No. 3).
 9. Plasmid, comprisinga hybrid nucleic acid sequence according to claim 6, associated with apromoter selected from the group consisting of the promoters which areconstitutively expressed and the promoters which are preferentiallyexpressed in the anthers, and with a terminator.
 10. Plasmid accordingto claim 9, wherein said sequence is under the control of the CaMV 35Spromoter and the terminator of the CaMV VI gene.
 11. Plasmid accordingto claim 9, comprising, in addition, at least one marker gene. 12.Transgenic plants, comprising in their nuclei an antisense hybridnucleic acid sequence, comprising, in the reverse direction, at leastthe same coding region of the unedited plant mitochondrial gene as thatcontained in the transgenic male-sterile plants according to claim 1.13. Plasmid, comprising a coding region of an antisense unedited plantmitochondrial gene, comprising, in the reverse direction, at least thesame coding region of unedited plant mitochondrial gene as thatcontained in the transgenic male-sterile plants according to claim 1,associated with a promoter selected from the group consisting ofpromoters which are constitutively expressed and promoters which arepreferentially expressed in the anthers, and with a terminator. 14.Process for producing male sterile transgenic plants comprisingtransforming a selected higher plant into a male sterile transgenicplant by introducing into a recipient plant at least one copy of thehybrid nucleic acid sequence that is capable of modifying male fertilityin plants having it incorporated therein, wherein said hybrid nucleicacid sequence comprises at least a coding region of an uneditedmitochondrial gene from a higher plant, with which is associated asequence capable of transferring the protein expressed by the saidcoding region to the mitochondrion, wherein: the coding region of theunedited mitochondrial gene is the wheat ATP6 gene in unedited form andligated to a mitochondrial transporter sequence, wherein the hybridnucleic acid sequence comprising the ATP 6 gene, when transformed into arecipient plant, causes male sterility; and wherein the nucleic acidsequence capable of transferring the said expressed protein to themitochondrion is selected from the group consisting of: the fragmentsencoding yeast tryptophanyl tRNA synthetase, the beta sub-unit ofNicotiana plumbaginifolia ATPase, the maize ATP/ADP translocator, and a303 base pair EcoRI/KpnI fragment comprising codons 1 to 62 of sub-unitIV of yeast cytochrome oxidase.
 15. Process for inhibiting theproduction of pollen in selected higher plants, comprising the followingsteps: (a) inserting a hybrid nucleic acid sequence that is capable ofmodifying male fertility in plants having it incorporated therein,wherein said sequence comprises at least a coding region of an uneditedmitochondrial gene from a higher plant, with which is associated asequence capable of transferring the protein expressed by the saidcoding region to the mitochondrion, wherein: the coding region of theunedited mitochondrial gene is the wheat ATP6 gene in unedited form andligated to a mitochondrial transporter sequence, wherein the hybridnucleic acid sequence comprising the ATP 6 gene, when transformed into arecipient plant, causes male sterility; and wherein the nucleic acidsequence capable of transferring the said expressed protein to themitochondrion is selected from the group consisting of: the fragmentsencoding yeast tryptophanyl tRNA synthetase, the β sub-unit of Nicotianaplumbaginifolia ATPase, the maize ATP/ADP translocator, and a 303 basepair EcoRI/KpnI fragment comprising codons 1 to 62 of sub-unit IV ofyeast cytochrome oxidase into the selected plants to form a transgenicplant of decreased male fertility; (b) regenerating and culturing thetransgenic plants obtained in (a); and (c) measuring the production andthe viability of pollen from said transgenic plants.
 16. Process forrestoring male fertility to transgenic male-sterile plants, comprisingthe following steps: (1) transforming a selected higher plant byintroducing at least one copy of a hybrid nucleic sequence that iscapable of modifying male fertility in plants having it incorporatedtherein, wherein said sequence comprises at least a coding region of anunedited mitochondrial gene from a higher plant, with which isassociated a sequence capable of transferring the protein expressed bythe said coding region to the mitochondrion, wherein: the coding regionof the unedited mitochondrial gene is the wheat ATP6 gene in uneditedform and ligated to a mitochondrial transporter sequence, wherein thehybrid nucleic acid sequence comprising the ATP 6 gene, when transformedinto a recipient plant, causes male sterility; and wherein the nucleicsequence capable of transferring the said expressed protein to themitochondrion is selected from the group consisting of: the fragmentsencoding yeast tryptophanyl tRNA synthetase, the β sub-unit of Nicotianaplumbaginifolia ATPase, the maize ATP/ADP translocator, and a 303 basepair EcoRI/KpnI fragment comprising codons 1 to 62 of sub-unit IV ofyeast cytochrome oxidase into a recipient plant, by means of a plasmidcontaining said sequence, whereby obtaining a transgenic male-sterileplant; (2) transforming the same higher plant as in (1), by introducingat least one copy of an antisense hybrid nucleic sequence comprising, inthe reverse direction, at least the same coding region of the uneditedplant mitochondrial gene as that contained in the said transgenicmale-sterile plants obtained in (1), into a recipient plant, by means ofa plasmid containing the said sequence, whereby obtaining transgenicmale-fertile plants; and (3) crossing the transgenic male-sterile plantsobtained in (1) and the male-fertile plants obtained in (2), in order toobtain hybrids.